Fluoresceinyl-ethylenediamine-ouabain detects an acidic environment in the cardiac glycoside binding site of Na+/K+-ATPase

Abstract

To probe the pH value in the microenvironment of the cardiac glycoside-binding site of Na+/K+ATPase, pH-sensitive fluorescent derivatives of ouabain were synthesized. The fluoresceinyl derivative of ethylenediamino-ouabain (FEDO) had a pK(s) of 6.0 and showed a H+-dependent fluorescence change, when its ratio of excitation at 490 nm/450 nm was recorded at 530 nm. Binding of FEDO inactivated Na+/K+-ATPase at 37°C and pH 7.25 in a slow time-dependent process under the conditions of backdoor phosphorylation with k(on) of 891 s-1 M-1. The complex dissociated with k(off) of 0.35x10-3 s-1 resulting in a K(d) value of 0.4 μM for the FEDO · enzyme complex. Binding of FEDO was associated with a decrease of the excitatory fluorescence ratio at 490 nm/450 nm which could be used to convert this change into a pH value. A pH value of 5.1±0.2 was calculated to exist in the microenvironment of the FEDO · enzyme complex. This pH value was independent of the pH of the incubation medium used to form the FEDO · enzyme complex. Analysis of the accessibility of the fluorophore in the FEDO · enzyme complex to the dynamic quencher potassium iodide detected a decrease of the Stern-Volmer constant from 6.2 mM-1 (free FEDO) to 1.5 mM-1 (FEDO · enzyme complex) indicating thereby a limited accessibility of the fluorophore to anions. Analysis of the microenvironment of the fluorescein residue of the FEDO · enzyme complex by measurements of the anisotropy and the fluorescence half-life time revealed that both processes differed significantly when H2O was replaced by D2O. We conclude, therefore, that a pH of 5.1 ± 0.2 exists in the vicinity of ouabain that is hidden in the depth of the receptor site when the ouabain receptor complex has been formed.